Treatment to obtain oncostasis



United States Patent 3,266,993 TREATMENT Ti) OBTAIN ONCOSTASIS Thomas J. Bardos, Snyder, N.Y., assignor, by mesne assignments, to Armour Pharmaceutical Company, a corporation of Delaware No Drawing. Filed July 26, 1961, Ser. No. 126,867 3 Claims. (Cl. 167-65) This invention relates to treatment in which there is obtained inhibition of neoplastic growth in animal studies and more particularly to treatment to obtain oncostasis with competitive antagonists of thymine and thymidine and inhibitors of the folic acid coenzyme systems.

This patent application is in part a continuation of my copending application Serial No. 847,678, filed October 21, 1959, now abandoned.

The treatment of this invention involves administering to a creature afilicted with neoplastic growth at least one compound of the group consisting of competitive antagonists of thymine and thymidine and at least one inhibitor of the folic acid coenzyme system. The term competitive antagonist refers to an analog of a compound which serves as a metabolite in a biosynthetic phenomenon by competing with the metabolite and preventing its access to substrate, which antagonism can be reversed by introducing into the biosynthetic system the immediate reaction product of such metabolite. Thus, this analog is an antimetabolite. Moreover, it will be understood that a suitable antimetabolite should not become a substitute metabolite in the biosynthetic system by being capable of only temporary inhibition of DNA biosynthesis and by ultimately being incorporated into the reaction product.

Although this treatment may be practiced by introducing into the DNA biosynthetic system, concomitantly, at least one compound from each of the aforementioned groups thereof, better results can be achieved by the administration to such biosynthetic system of a composition including at least one compound of the group consisting of competitive antagonists for thymine and thymidine and at least one inhibitor of the folic acid coenzyme system.

Thus, at least one compound from each of these groups of reactants can be incorporated into a suitable vehicle, and the resulting composition may be introduced into a system in which there is active biosynthesis of DNA to accomplish the purposes of this invention.

One class of compounds which may be employed in this invention as the competitive antagonist of thymine or thymidine can be illustrated by the formulae:

wherein X is selected from the group consisting of II II I! and S, wherein R is a monovalent organic radical selected w 3,266,993- lca Patented August 16, 1966 from the group consisting of amino, uracil and alkyl having less than 4 carbon atoms, and wherein R is selected from the group consisting of hydrogen, ribosyl and desoxyribosyl.

These compounds can be further characterized as being degradable to S-mercaptouracil by hydrolysis, dissociation, oxidation, reduction or transmethylation. That is, these compounds are convertible to S-mercaptouracil by reductive hydrolysis, which reaction refers to hydrolysis under reducing conditions. In a practical sense, these compounds are characterized by being capable of degradation to S-mercaptouracil in aqueous solution having a pH of from 9 to 13 and containing one mole equivalent of glucose although more precise control is achieved at a pH Within the range of 9.5 to 10.5 and at a temperature of from to C. at atmospheric pressure. It will be understood that the degradation product obtained in this reaction is the alkali salt of 5-mercaptouracil, and that such alkali salt may be converted to free S-mercaptouracil by acidification.

It will also be understood that S-mercaptouracil, i.e., the compound in the foregoing formulae on the left hand, in which R represents hydrogen 'and the riboside and deoxy riboside thereof, can be obtained in an alkaline reaction medium as the alkali salt, and consequently that such alkali salts are deemed to be included in this class of compounds, i.e., wherein the hydrogen in the 5 position of the left hand formula represents a metal such as zinc, sodium, etc.

Although S-mercaptouracil and the riboside and deoxyriboside thereof represent especially desirable compounds for the purposes of this invention, there may also be employed those compounds having the left hand formula above in which the hydrogen in the 5 position is replaced by chlorine. In addition, other compounds represented by the foregoing formulae which may be employed in this invention, are, as follows: S-uracilylethylxanthate, i.e., wherein X represents S-uracilyldisulfide, i.e., wherein X represents S; uracil-5- isothiouronium chloride, i.e., wherein X represents lfiIH o and S-acetyl-S-mercaptouracil, wherein X represents It will be apparent that the utility of these compounds for the purposes of this invention is predicated on the analogous relationship of the S-thio function of the molecule to thymine and thymidine, and thus the substituent X in this formula is such that it is subject to cleavage in physiological systems. Consequently, it will be seen that the monovalent organic radical described in connection with the formulae is not functionally involved in the utilitarian aspects of this invention and that consequently there may be substituted thereon any monovalent organic radical.

Certain of these thymine and thymidine antagonists, and the method of the preparation thereof, are described in the co-pending patent application of T. J. Bardos et al.,

Serial No. 578,197, filed April 16, 1956, and in my copending patent application Serial No. 772,721, filed November 10, 1958, both now abandoned.

The term inhibitor of the folic acid coenzyme system refers to an antagonist to active forms of folic acid, such as inhibitors of folic acid, tetrahydrofolic acid, folinic acid and cofactor F.

One class of compounds which may be employed in this invention as the inhibitor for the folic acid coenzyme system can be illustrated by the formulae:

N BIN/T R HN: R

wherein X is either an imine or an oxygen group, wherein R is an organic group having steric resemblance to a methyl radical, e.g. methyl, ethyl, or halogen, and wherein R is either an alkyl or substituted alkyl radical. The compounds represented by Formulae A1 and A2 may contain either an imino or oxygen group attached at the 2 and 4 positions, but such compounds should contain at least one of such imino groups. Since the imino or oxygen group attached at the 2 and 4 positions of these formulae are in an enolizable form, it will be understood that these formulae represent also desmotropes of these compounds.

The compounds of the foregoing Formulae A1, A2 and B have been found to be antagonists of riboflavin, as well as being inhibitors of the folic acid coenzyme system, especially in being antagonists for folinic acid.

The class of compounds represented Formulae A1 and A2 are related to isoalloxazine. When R in such Formulae A1 and A2 is a methyl radical and X is an imino group, the class of compounds represented thereby may be designated as 2,4 diimino 2,4 desoxy 6,7 dimethyl 9 substituted isoalloxazines. Further, when R in Formulae A1 and A2 is a ribityl radical, the compound is 2,4 diimino 2,4 desoxy riboflavin. Also, when R in these formulae is methyl, the compound may be designated as 2,4 diimino 2,4 desoxy lumiflavin. On the other hand, when X in Formulae A1 and A2 is a hydroxyl group, the compounds represented are the corresponding 2 imino 2 desoxy 6,7 disubstituted 9 substituted isoalloxazine and 4 imino 2- desoxy 6,7 di substituted 9 substituted isoalloxazine, respectively, in which the substituents at the 6 and 7 positions are either methyl, ethyl or halogen. An especially desirable class of compounds results when the substituent represented by R in Formulae A1 and A2 is an alkyl or substituted alkyl radical containing not more than 6 carbon atoms, and an especially desirable class of compounds results when R is a methyl, ethyl, ribityl or ribosyl radical.

The class of compounds represented by Formulae A1 and A2 may be prepared by aminating a chlorinated riboflavin analog of the structure;

wherein X is either oxygen or a dichloro group, wherein R is a group having steric resemblance to a methyl radical, e.g., methyl, ethyl or halogen, and where R is either an alkyl or a substituted alkyl radical. The steric configuration of the compounds represented by this formula requires a distribution of 3 chlorine atoms between the 2 and 4 positions. Thus, either the 2 or 4 position may have a dichloro substituent. In the preferred practice of this method, riboflavin may be acetylated with acetic anhydride in a pyridine medium. The resulting tetracetyl derivative of riboflavin may be chlorinated with phosphorus pentachloride in an appropriate solvent, such as phosphorus oxychloride, phosphorus trichloride, chloroform, etc. This chlorinated compound can be aminated with such ammonia reactants as alcoholic ammonia, liquefied ammonia, concentrated aqueous ammonia, etc., and then de-acetylated to obtain the riboflavin analog. The chlorination of the tetracetyl derivative of riboflavin may be such as to produce either the 2 chloro 4 oxy, 4 chloro 2 oxy, or 2,4 trichloro compound. The respective chlorine derivative can be aminated and de-acetylated to produce the corresponding analog of riboflavin.

The compounds represented by Formula B may be described as analogs of alloxazine, and when X in such Formula B represents an imino group the resulting compounds may be termed 2,4-diimino-2,4-desoxy-6,7-di-substituted alloxazines, and when R is a methyl radical the compound is 2,4-diimino-2,4-desoxy lumichrome. Also, when X in Formula B is an oxygen atom the compounds represented thereby are 2-imino-2-desoxy-6,7-di-substituted alloxazines, and the corresponding 6,7-dimethyl compound is 2-imino-2-desoxy lurnichrome.

These antagonists of both folinic acid and riboflavin, and the preparation thereof, are described in T. I. Bardos and D. B. Olsen US. Patent No. 2,867,614.

Another class of inhibitors of the folic acid coenzyme system are the anti-folic acid compounds of which the most widely known member is aminopterin. Another example of this class of compounds is amethopterin.

The concomitant and simultaneous utilization of these two classes of compounds in the inhibition of DNA biosynthesis is predicated upon a synergism in which inhibition is achieved with a concentration of a mixture of such compounds significantly less than that with which such inhibition may be achieved with either compound alone. That is, S-mercaptouracil and derivatives thereof have been found to be relatively ineffective in certain DNA biosynthetic systems. On the other hand, 2,4-diimino-6,7-dimethyl 2,4 deoxyalloxazine and derivatives thereof and aminopterin, although effective in inhibiting certain DNA biosynthetic systems, are significantly in creased in effectiveness in inhibiting DNA biosynthesis in combination with S-mercaptouracil and derivatives thereof. It is postulated that in certain DNA biosynthetic systems there are alternative pathways for the production of DNA, and that a single compound may inhibit only one of these alternative pathways and DNA biosynthesis may continue through the alternative pathway. It is believed that the 2,4-diimino-6,7-dirnethyl-2,4-deoxyalloxazine and derivatives thereof and aminopterin are eifective in inhibiting the synthesis of DNA by one alternative pathway, while S-mercaptouracil and derivatives thereof are effective in inhibiting the biosynthesis of DNA by another pathway. Consequently, the utility of this invention is predicated upon the observed inhibition of DNA biosynthesis by concomitant or combined introduction therein of at least one compound of the 2,4-diimino-6,7-dimethyl- 2,4-deoxyalloxazine or aminopterin series and at least one compound of the S-mercaptouracil series.

Accordingly, it has been discovered that the concomitant and simultaneous administration of at least one of the thymine antagonists and at least one of the inhibitors of the folic acid coenzyme system to a creature afilicted with a neoplasm provides selective inhibition of neoplastic growth greater than can be achieved with a compound of either of these series alone. Consequently, treatment of this invention is useful in obtaining oncostasis in creatures.

It will be apparent that the treatment of this invention can be carried out by a concomitant or simultaneous administration of at least one of each of the two groups of compounds. That is, in the practice of this invention, there may be administered to a creature afflicted with neoplastic growth at least one of each of these two groups of compounds at different time intervals in the course of treatment. On the other hand, in practicing the treatment of this invention, it is preferred to employ a combination of at least one of each of these two groups of compounds administered simultaneously to a creature afilicted with neoplastic growth.

Thus, the practice of this invention may involve the utilization of one compound of each of the two groups in a pharmaceutical carrier such as normal saline, each administered at different time intervals in the course of treatment. However, as suggested hereinabove, it is preferred to employ a combination of at least one of each of these two groups of compounds in a single pharmaceutical carrier such as normal saline, and to administer the resulting composition to the creature afilicted with neoplastic growth.

This invention can be further illustrated by the following specific examples:

Example I The following is a comparison of the effect on mouse sarcoma 180 by (1) S-mercaptouracil, (2) 2,4-diimino- 6,7-dimethyl-2,4-deoxyalloxazine, and (3) a combination of the above compounds:

Groups of iCR/Ha Swiss male mice, mice per group, were treated by the subcutaneous injection of 10x10 sarcoma 180 cells. Treatment with the foregoing compounds was started 24 hours after the injection of the sercoma 180 cells, and such treatment was suspended after 7 days. The animals were sacrificed 14 days after the injection of sarcoma 180 cells, and the tumors thereof were weighed.

The results were as follows:

Mean Tumor Treatment (mg/kg.) Wei ht (mg) No. Died The following is a comparison of the effect on leukemia Ll2l0 in DBA/2 mice by (l) S-mercaptouracil, (2) amethopterin, and (3) a combination of the above.

Groups of DBA/2 mice, 10 mice per group, were treated by the subcutaneous inoculation of 10 10 leukemia Ll210 cells. Treatment was instituted by the intraperitoneal administration of the foregoing compounds 24 hours after the inoculation of the leukemia cells. This treatment was continued for five days. The mice were sacrificed 10 days after inoculation with the leukemia cells, and the tumors thereof were weighed.

The results were as follows:

No. of Treatment (mg/kg.) No. Mean Tumor Mice Died Weight (mg) Without Tumor The foregoing results demonstrate that whereas 5- mercaptouracil had little or no effect in effecting the regression of leukemia LlZlO tumors, and whereas amethopterin has a definite effect in bringing about regression of leukemia Ll210 tumors, the combination of S-mercaptouracil and amethopterin provides significant regression of leukemia Ll210 tumors.

Example 111 The following comparison between (1) S-mercaptouracil, (2)amethopterin, and (3) a combination of the above was carried out in a manner similar to that of Example II, except that treatment was effected by the subcutaneous administration of such compounds.

No. of

Treatment (mg/kg.) N0. Mean Tumor Mice Died Weight trng.) Without Tumor 1 Animals without tumors were not included in the group mean weight Example IV Mean Survival Time (Days) Treatment (mg/kg.)

Control 7 6 1 (50) 7. 4 5. 8 s 6.4

The foregoing results demonstrate that whereas either 5-mercaptouracil or amethopterin have little or no effect in prolonging the survival time of mice afllicted with leukemias 49468 and 4946R, the combination of S-mercaptouracil and amethopterin significantly prolongs the survival time of mice afilicted with leukemia 49468, but

7 little effect is obtained with such combination on leukemia 4946R.

It will be apparent in view of the foregoing that this composition is useful as an on-costatic agent.

While in the foregoing specification this invention has been described in considerable detail by reference to specific embodiments thereof, it will be apparent to those skilled in the art that this invention is susceptible to other embodiments and that many of the details can be varied widely, without departing from the basic concept and spirit of the invention.

I claim:

(1. A composition in dosage unit form containing, as essential active ingredients, neoplastic growth inhibiting quantities of at least one compound having the formulae and and S wherein R is a monovalent organic radical selected from the group consisting of amino, uracil and alkyl having less than 4 carbon atoms, and wherein R is selected from the group consisting of hydrogen, ribosyl and deoxyribosyl; at least one compound having the formulae wherein X is selected from the group consisting of oxygen and an imino group, wherein R is selected from the group consisting of methyl, ethyl and halogen, wherein R is a radical of the group consisting of alkyl and hydroxyalkyl radicals, and wherein at least one X in Formula A is inimo; and pharmaceutically acceptable excipients.

2. A composition in dosage unit form containing neoplastic growth inhibiting quantities of S-mercaptouracil and 2,4-diminio-6,7-dimethyl-2,4-deoxyalloxazine as essential active ingredients, and pharmaceutically accept able excipients.

3. A composition in dosage unit form containing neoplastic growth inhibiting quantities of S-meroaptouracil and amethopterin as essential active ingredients, and pharmaceutically acceptable excipients.

References Cited by the Examiner UNITED STATES PATENTS 1/1959 Bardos et al.

OTHER REFERENCES LEWIS GOTTS, Primary Examiner.

MORRIS O. WOLK, Examiner.

DONALD LEVY, Assistant Examiner. 

1. A COMPOSITION IN DOSAGE UNIT FORM CONTAINING, AS ESSENTIAL ACTIVE INGREDIENTS, NEOPLASTICE GROWTH INHIBITING QUANTITIES OF AT LEAST ONE COMPOUND HAVING THE FORMULAE 